<i>Vatairea guianenis</i> Aubl. (Fabaceae) is an Amazonian medicinal plant species traditionally used for treating skin diseases. In an initial screening, a <i>V. guianensis</i> leaf extract and its subextracts showed antibacterial and antifungal activities. The EtOAc subextract was selected for chemical workup and afforded five known (<b>1</b>-<b>4</b> and <b>8</b>) and six undescribed isoflavones, vatairenones C-H (<b>5</b>-<b>7</b> and <b>9</b>-<b>11</b>). All isoflavones are prenylated in position C-8, displaying either chain-prenylated (<b>1</b>-<b>7</b>) or ring-closed forms (<b>8</b>-<b>11</b>). The most bioactive compound (<b>3</b>) exhibited <i>in vitro</i> activity against clinically relevant bacteria and fungi with IC<sub>50</sub> values ranging from 6.8 to 26.9 μM. Due to its broad antimicrobial activity and low general toxicity, compound <b>3</b> is a potential lead compound for structural modifications. The results of the present study support the ethnomedicinal use of <i>V. guianensis</i> in the treatment of dermatological disorders. <sup>1</sup>H NMR spectra of some of the isolated compounds showed intricate signal patterns, which might explain repeated errors in assigning the correct structure of the isoflavonoid B-ring in the literature and which we resolved by higher order spectra simulations.